Preparation and composition of inter-alpha inhibitor proteins from human plasma for therapeutic use

ABSTRACT

The invention relates to Inter-alpha inhibitor proteins (IαIp). The invention further relates to processes for purification of IαIp compositions and their use for treatment of human diseases such as sepsis and septic shock, rheumatoid arthritis, cancer and infectious diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a reissue of U.S. Pat. No. 7,932,365, which is aU.S. national phase application, pursuant to 35 U.S.C. §371, of PCTinternational application Ser. No. PCT/US04/036848 filed Nov. 5, 2004,designating the United States and published in English on May 26, 2005as publication WO 2005/046587 A2, which claims priority to U.S.provisional applications application Ser. No. 60/518,366, filed Nov. 8,2003, and Ser. No. 60/617,166, filed Oct. 8, 2004. The entire contentsof the aforementioned patent and patent applications are incorporatedherein by this reference.

RELATED APPLICATION

This application contains subject matter that is related to thatdisclosed in provisional patent application Ser. No. 60/518,366 filedNov. 8, 2003, entitled, “Preparation and Composition of Inter-alphaInhibitor Proteins from Human Plasma for Therapeutic Use,” thedisclosure of which application is incorporated herein in its entiretyby this reference.

GOVERNMENT SUPPORT

A portion of this invention may have been supported by NationalInstitutes of Health grants RO1 GM053008, R01 GM057468, and R43GM065667.STATEMENT AS TO FEDERALLY FUNDED RESEARCH

This invention was made with government support under Grant Nos. R01GM053008, R01 GM057468, and R43 GM065667 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

The inter-alpha inhibitor protein (IαIp) family is a group ofplasma-associated serine protease inhibitors. Members of this family arecomposed of heavy and light polypeptide subunits that are covalentlylinked by a glycosaminoglycan. The light chain, also called bikunin, isresponsible for the serine protease inhibitory activity of themolecules. The name “bikunin” reflects the presence of 2protease-inhibiting domains of the Kunitz type. In normal plasma,bikunin is found mostly in a complex form as inter-alpha inhibitor(IαI), which has a molecular weight of 225 kDa, and pre-alpha inhibitor(PαI), which has molecular weight of 120 kDa. In IαI, bikunin is linkedto 2 heavy polypeptide chains, H1 and H2, whereas, in PαI, only a singleheavy chain (H3) is linked to bikunin. In these complexed forms, bikuninremains inactive until its release by partial proteolytic degradation, amechanism that serves as a means to regulate activity. After cleavagefrom the complex, the activated bikunin is cleared rapidly from plasmaby glomerular filtration, a process that is facilitated by its lowmolecular weight and by receptor-mediated uptake. U.S. Pat. Nos.6,489,128 and 6,660,482 are related to the use of diagnosing cancer andsepsis, respectively. Methods of inhibiting metastases and of treatingsepsis are also disclosed, however, the compositions were notsubstantially pure and had stability, i.e., short half-lives, problems.

Despite the introduction of antibiotics over fifty years ago, whichindeed saw a decline of sepsis-induced mortality from 55% to 35%,medicine has not benefited from a significant reduction in mortality ofsubjects with sepsis. In fact, sepsis continues to be one of the leadingcauses of death in intensive care units and a large number of septicsubjects die of ensuing septic shock and multiple organ failure. Sepsisis a systemic response to infection, e.g., a bacterial infection. It iscommonly caused by endotoxins from Gram negative bacteria or exotoxinsfrom Gram positive bacteria (which can trigger endotoxin-likeresponses). The systemic response can lead to septic shock, which ischaracterized by a precipitous drop in blood pressure, cardiovascularcollapse, and/or multiple organ failure. The mortality rate amongsubjects diagnosed with septic shock can be as high as 35-45%. Rapidlyand reliably treating sepsis has been difficult using conventionalmedications.

Sepsis and septic shock are associated with activation of innateimmunity and coagulation systems. Sepsis and septic shock arecharacterized clinically by systemic inflammation, coagulopathy,hypotension and multiple organ dysfunction (J.-L. Vincent et al.,Annuals of Medicine 34 (2002) 606-613). During severe sepsis, a networkof specific proteases activates clotting, fibrinolytic and complementfactors. These proteases can also trigger tissue and organ damage andenhance non-specific proteolysis of clotting and complement factors inplasma (J. Wite et al., Intensive Care Medicine 8 (1982) 215-222; S. J.Weiss, New England Journal of Medicine 320 (1989) 365-376).

BRIEF SUMMARY OF THE INVENTION

The present invention relates to a process for the purification ofinter-alpha inhibitor proteins (IαIp) from human plasma and their usefor treatment of human diseases such as sepsis, acute inflammatorydiseases, severe shock, septic shock, rheumatoid arthritis, cancer,cancer metastasis, infectious diseases, and preterm labor; or reducingthe risk of mortality associated with sepsis, acute inflammatorydiseases, severe shock, septic shock, rheumatoid arthritis, cancer,cancer metastasis, infectious diseases, and preterm labor.

According to one aspect, a process for producing a blood plasma-derivedIαIp composition, wherein IαI and PαI are present in the mixture in aphysiological proportion comprises isolating from blood plasma a plasmafraction containing IαI and PαI, wherein the IαI and PαI are present ina physiological proportion; and purifying the plasma fraction to obtainan IαIp composition with a purity of IαIp ranging from about 85% toabout 100% pure.

According to another aspect a composition of IαIp comprises a mixture ofinter-alpha inhibitor protein (IαI) and pre-alpha protein (PαI), whereinthe IαI and the PαI are present in said mixture in a physiologicalproportion ranging from about 85% to about 100% pure.

In a related aspect, a composition of IαIp comprises a mixture ofinter-alpha inhibitor protein (IαI) and pre-alpha protein (PαI), whereinthe IαI and the PαI are present in said mixture in a physiologicalproportion and having a high trypsin inhibitory specific activity.

In another related aspect, a composition of IαIp comprises IαI and PαIhaving a half-life of greater than one hour.

In yet another related aspect, a composition of IαIp comprises IαI andPαI wherein the IαI and PαI are composed of a light chain of inter-alphainhibitor protein associated with at least one of three heavy chains H1,H2 and H3.

In another related aspect, a composition of IαIp comprises a mixture ofinter-alpha inhibitor protein (IαI) and pre-alpha protein (PαI), whereinthe IαI and the PαI are present in the mixture in a physiologicalproportion comprising a light chain of inter-alpha inhibitor proteinassociated with at least one of four heavy chains-H1, H2, H3 and H4.

In still another related aspect, a composition of IαIp is made accordingto the process, which comprises isolating from blood plasma a plasmafraction containing IαI and PαI, wherein the IαI and PαI are present ina physiological proportion; and purifying the plasma fraction to obtainan IαIp composition with a purity of IαIp ranging from about 85% toabout 100% pure.

In another aspect, a pharmaceutical composition according to theinvention comprises a therapeutically effective composition of IαIp asdescribed herein and a pharmaceutically acceptable carrier.

Another aspect relates to a method of treating an inflammation relateddisorder, cancer, or an infectious disease in a subject, which comprisesadministering a therapeutically effective amount of IαIp produced by anyof the processes described infra.

In another related aspect, a method of treating a subject comprisesdetermining the pre-treatment level of one or more of IαI, PαI, IαIp,H3, H4, H1, H2, and LC; and administering a therapeutically effectiveamount of IαIp to the subject.

In a related aspect, a method for predicting a response to an IαIptherapy is described. The method comprises assaying a sample obtainedfrom a subject to detect the level of one or more of IαI, PαI, IαIp, H3,H4, H1, H2, and LC; wherein the detected levels identifies a subjectthat may respond favorable to IαIp therapy.

In another related aspect, a method of monitoring the progress of asubject being treated with an IαIp therapy is described and comprisesdetermining the pre-treatment level of one or more of IαI, PαI, IαIp,H3, H4, H1, H2, and LC; administering a therapeutically effective amountof IαIp to the subject; and determining the level of one or more of thelevels in the subject after an initial period of treatment with theIαIp, wherein an increase of the level in the subject followingtreatment with IαIp indicates that the subject is likely to have afavorable clinical response to treatment with IαIp.

In another aspect, a kit for IαIp therapy is described and comprises oneor more of IαI, PαI, IαIp, H3, H4, H1, H2, and LC; and instructions fortherapeutic use.

In a related aspect, a composition is described, which comprises acontainer including IαIp and a label or package insert with instructionsfor administering the IαIp to a subject.

In a further related aspect, a kit is described, which comprises acomposition as described above as well as instructions for therapeuticuse.

Other embodiments of the invention are disclosed infra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts histopathology of the spleen. Penicillary arterysurrounded by preserved white pulp with extensive loss of red pulpcellular elements in the control animal (A) versus in the IαIp-treatedanimal (B) with normal surrounding white pulp and slightly hypercellularred pulp (H&E stain, magnification 20×).

FIG. 2 depicts the amino acid sequence of H4 (SEQ ID NO: 1).

FIG. 3 depicts the alterations in bikunin mRNA expression in the liverat 5 and 20 h after cecal ligation and puncture (CLP) or sham operation(Sham). Data are expressed as means ±SE (n=8/group) and compared byone-way analysis of variance (ANOVA) and Tukey's test: * P<0.05 versusshams. FIG. 3a shows the mRNA amplified and separated by size on a gel,and FIG. 3b graphically depicts the results with the bikunin levelsbeing normalized to the G3PDH expression.

FIG. 4 depicts the alterations in the t_(1/2) of 125I-IαI at 5 and 20 hafter cecal ligation and puncture (CLP) or sham operation (Sham). Dataare expressed as means ±SE (n=5/group) and compared by one-way analysisof variance (ANOVA) and Tukey's test:

FIG. 5 depicts the alterations in the survival rate at 10 days aftercecal ligation and puncture and cecal excision with vehicle treatment(CLP+Vehicle) and cecal ligation and puncture with inter-α-inhibitortreatment (CLP+IαIp). There were 12 animals in each group. The survivalrate was estimated by the Kaplan-Meier method and compared by using thelog-rank test * P<0.05 vs. CLP+Vehicle.

FIG. 6 depicts the alterations in the survival rate at 10 days aftercecal ligation and puncture and cecal excision with vehicle treatment(CLP+Vehicle) and cecal ligation and puncture with inter-α-inhibitortreatment (CLP+IαIp). There were 11 to 12 animals in each group. Thesurvival rate was estimated by the Kaplan-Meier method and compared byusing the log-rank test. * P<0.05 vs. CLP+Vehicle.

FIG. 7 describes alterations in the survival rate at 10 days after cecalligation and puncture and cecal excision with vehicle treatment(CLP+Vehicle) and cecal ligation and puncture with inter-α-inhibitortreatment (CLP+IαIp). There were 16 animals in each group. The survivalrate was estimated by the Kaplan-Meier method and compared by using thelog-rank test. * P<0.05 vs. CLP+Vehicle.

FIGS. 8a and 8b describe purification schemes of IαIp from human plasma,according to the invention.

FIG. 9 graphically depicts the beneficial effects of highly purifiedIαIp in the animal studies of sepsis.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein is a new process for purifying IαIp from plasma.Further disclosed herein is a therapeutic composition of purified IαIpfor administration to a subject to treat acute inflammatory disease,sepsis, severe shock, septic shock, rheumatoid arthritis, cancer, cancermetastasis, infectious disease, and preterm labor.

Prior known purifications contained contamination by factor X (FX),which is detected as an 80 kDa band by Western blot analysis. The FX wasalso detected in a clotting assay. The removal of FX is importantbecause FX is considered thrombogenic and can be harmful if administeredto humans. The methods described herein solve the FX contaminationproblems of previous IαIp compositions.

Inter-alpha inhibitor proteins (IαIp) are a family of structurallyrelated serine protease inhibitors found at relatively highconcentrations (400-800 mg/L) in human plasma. IαIp is a large,multi-component complex that functions as a trypsin-type proteaseinhibitor. Unlike other inhibitor molecules, this family of inhibitorsconsists of a combination of polypeptide chains (light and heavy chains)covalently linked uniquely by a chondroitin sulfate chain. The heavychains of Inter-alpha proteins (H1, H2 and H3) are also calledHyaluronic acid (HA) binding proteins. The major forms found in humanplasma are inter-alpha-inhibitor (IαI), which consists of two heavychains (H1 & H2) and a single light chain (L), and pre-alpha-inhibitor(PαI), which consists of one heavy (H3) and one light chain (L). Thelight chain (also termed bikunin (bi-kunitz inhibitor, having two Kunitzdomains) is known to broadly inhibit plasma serine proteases. Thecomplex has been shown to be important in the inhibition of an array ofproteases, including neutrophil elastase, plasmin, trypsin,chymotrypsin, cathepsin G, and acrosin.

IαI and PαI have also been found to be complexed with H4, another heavychain of IαIp proteins. Certain embodiments of IαIp compositionsaccording to the invention contain H4 in complex with PαI, IαI, or bothPαI and IαI.

Without wishing to be bound by any scientific theories, we speculatethat heavy chains of IαIp, after being released from the complex, bindHA preventing HA from binding its receptor, CD44. In the absence ofheavy chains of IαIp, HA will bind to CD44 and trigger the secretion ofpro-inflammatory factors, for example, TNF-alpha, and causeinflammation. Meanwhile, the light chains of IαIp, once released fromthe complex exhibit anti-protease activity.

“IαIp composition” refers to a preparation of IαIp proteins, includingIαI and PαI in physiological proportions. Physiological proportions, asused herein is intended to include proportions found in a person oranimal that is not suffering from an infection or condition, and/or theratio of IαI to PαI that appears naturally in human plasma.Physiological proportions are usually from between about 60% to about80% IαI and between about 40% to about 20% PαI. Physiologicalproportions may vary from these ranges due to normal variations ingenetic makeup of subjects.

As used herein, “mixture of inter-alpha inhibitor protein (IαI) andpre-alpha protein (PαI)” refers to a composition containing both the IαIand PαI complexes. The mixture may also contain buffers, salts, or othercomponents that are used to isolate the IαIp complex. In certainaspects, the IαI and the PαI are present in the mixture in aphysiological proportion.

IαI and PαI present in the plasma fraction have an apparent molecularweight of between about 60,000 to about 280,000 60 to about 280 kDa.Molecular weight may be determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis.

“Half-life,” as used herein, refers to half of the amount of time thatthe administered IαIp is active upon administration. IαIp compositionsaccording to the invention have a half-life of, for example, greaterthan about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 7.5, or 10 hours. In apreferred embodiment, the IαIp composition has a half-life of greaterthan about 5 hours. In a particularly preferred embodiment, the IαIpcomposition has a half-life of greater than about 10 hours. Longerhalf-lives are preferred, for example, because fewer doses are requiredto be administered to a subject over time.

The IαIp compositions of the invention may have a high trypsininhibitory specific activity. The trypsin inhibitory specific activityof the IαIp compositions according to the invention may range frombetween about 1000 to about 2000 IU/mg. Preferably the trypsininhibitory specific activity is above 1200 IU/mg and even morepreferably above 1500 IU/mg. The trypsin inhibitory specific activitymay be measured, for example, by the trypsin inhibitory assay usingL-BAPA as a substrate. See, H U Bergmeyer, ed: vol 5, 3^(rd) ed. 119(1984) Verlag Chemie, Weinheim: Chromogenic substrate for the assay oftrypsin: R. Geiger, H. Fritz, Methods of Enzymatic Analysis.

A composition of IαIp may be a mixture of inter-alpha inhibitor protein(IαI) and pre-alpha protein (PαI), wherein the IαI and the PαI arepresent in said mixture in a physiological proportion comprising a lightchain of inter-alpha inhibitor protein associated with at least one ofthree heavy chains H1, H2 and H3. A composition according to theinvention may also have a light chain of inter-alpha inhibitor proteinassociated with at least one of four heavy chains H1, H2, H3 and H4.Examples of each protein in the IαIp complex are as follows: BikuninGenBank accession number: AAB84031, P02760; H1 GenBank accession number:P19827, NP_002206; H2 GenBank accession number: NP_002207, P19823; H3GenBank accession number: NP_002208; H4 GenBank accession number:Q14624, NP_002209, which are each incorporated herein by reference intheir entirety.

As used herein “blood plasma-derived” refers to being originallyisolated or purified from blood plasma. That is, the natural environmentof the composition is blood plasma.

As used herein, “a plasma fraction” is a fraction from an isolation orpurification step, for example, chromatography, that was originallyderived from blood plasma. Plasma fractions according to the inventionmay be for example, a side fraction obtained from the purification ofclotting factor IX, a side fraction from the purification of aprothrombin complex concentrate, a cryosupernatant resulting fromcryoprecipitation (described in Hoffer et al., Journal of ChromatographyB 669 (1995) 187-196) of blood plasma, or cryo-poor plasma cryo-poorplasma is used interchangeably with cryosupernatant herein. Thecryo-poor plasma is the supernatant obtained from cryoprecipitation.

An example of a side fraction according to the invention is one obtainedfrom the purification of clotting factor IX. A mixture of IαI/PαI hasbeen shown to be present in side-fractions generated during thepurification of factor IX (FIX). The method of obtaining the sidefraction obtained from the purification of clotting factor IX isdescribed in Hoffer et al., Journal of Chromatography B 669 (1995)187-196, which is hereby incorporated by reference in its entirety.Other examples of side fractions include a side fraction from FIXpurification or a side fraction from the purification of a prothrombincomplex concentrate, as is described in D. Josic et al., ThrombosisResearch 100 (2000) 433-441, which is hereby incorporated by referencein its entirety; and a side fraction isolated as a cryosupernatantresulting from cryoprecipitation of blood plasma. (For example, suitablecryoprecipitation methods are described in Hoffer et al., Journal ofChromatography B 669 (1995) 187-196.) Other plasma fractions that may beuseful to purify IαIp complexes from blood include strong anion-exchangefractions and monolith chromatographic fractions, which are describedbelow in the examples.

Plasma fractions, according to the invention, may be from human,primate, bovine, porcine, feline, or canine sources.

As used herein, the term “obtaining” includes purchasing, synthesizing,isolating or otherwise acquiring one or more of the substances used inpracticing the invention.

Thus, “obtaining blood,” as used herein, includes acquiring blood, forexample, from human, primate, bovine, porcine, feline, or caninesources. The blood may be acquired and/or purchased from, for example,blood banks, hospitals, hospices, private companies, researchfoundations, or any other source of blood.

“Obtaining blood plasma,” as used herein, includes acquiring bloodplasma, for example, from human, primate, bovine, porcine, feline, orcanine sources. The blood plasma may be acquired and/or purchased from,for example, blood banks, hospitals, hospices, private companies,research foundations, or any other source of blood. Alternately, bloodplasma may also be isolated from blood once blood is obtained. Suitablemethods of isolating blood plasma include gravity and centrifugation.

As used herein, “obtaining a side fraction obtained from thepurification of clotting factor IX,” includes acquiring and/orpurchasing side fractions obtained from the purification of clottingfactor IX from for example, a company or hospital that routinelypurifies factor IX.

As used herein, “obtaining a side fraction from the purification of aprothrombin complex concentrate,” includes acquiring and/or purchasingthe side fractions, for example, from a company, research organizationor hospital that purifies prothrombin complex.

As used herein, “obtaining a cryosupernatant resulting fromcryoprecipitation of blood plasma,” includes acquiring and/or purchaseda cryosupernatant, for example, from a hospital, research organization,or company that cryopercipitates blood plasma in a manner suitable foruse in the invention.

“Solid support” refers to a solid material which can be derivatizedwith, or otherwise attached to, a capture reagent. Exemplary solidsupports include probes, microtiter plates and chromatographic resins.

“Adsorption” refers to detectable non-covalent binding of an analyte toan adsorbent or capture reagent. An adsorbent surface refers to asurface to which is bound an adsorbent (also called a “capture reagent”or an “affinity reagent”). An adsorbent is any material capable ofbinding an analyte (e.g., a target polypeptide or nucleic acid).

A chromatographic adsorbent refers to a material typically used inchromatography. Chromatographic adsorbents include, for example, ionexchange materials, metal chelators (e.g., nitriloacetic acid oriminodiacetic acid), immobilized metal chelates, hydrophobic interactionadsorbents, hydrophilic interaction adsorbents, dyes, simplebiomolecules (e.g., nucleotides, amino acids, simple sugars and fattyacids) and mixed mode adsorbents (e.g., hydrophobicattraction/electrostatic repulsion adsorbents).

A biospecific adsorbent refers to an adsorbent comprising a biomolecule,e.g., a nucleic acid molecule (e.g., an aptamer), a polypeptide, apolysaccharide, a lipid, a steroid or a conjugate of these (e.g., aglycoprotein, a lipoprotein, a glycolipid, a nucleic acid (e.g.,DNA)-protein conjugate). In certain instances the biospecific adsorbentcan be a macromolecular structure such as a multiprotein complex, abiological membrane or a virus. Examples of biospecific adsorbents areantibodies, receptor proteins and nucleic acids. Biospecific adsorbentstypically have higher specificity for a target analyte thanchromatographic adsorbents.

“Eluent” or “wash solution” refers to an agent, typically a solution,which is used to affect or modify adsorption of an analyte to anadsorbent surface and/or remove unbound materials from the surface. Theelution characteristics of an eluant can depend, for example, on pH,ionic strength, hydrophobicity, degree of chaotropism, detergentstrength and temperature.

“Analyte” refers to any component of a sample that is desired to bedetected. The term can refer to a single component or a plurality ofcomponents in the sample.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues are analogs or mimetics of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers.Polypeptides can be modified, e.g., by the addition of carbohydrateresidues to form glycoproteins. The terms “polypeptide,” “peptide” and“protein” includes glycoproteins, as well as non-glycoproteins.

“Immunoassay” is an assay that uses an antibody to specifically bind anantigen (e.g., an IαIp complex). The immunoassay is characterized by theuse of specific binding properties of a particular antibody to isolate,target, and/or quantify the antigen.

“Antibody” refers to a polypeptide ligand substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof, whichspecifically binds and recognizes an epitope (e.g., an antigen). Therecognized immunoglobulin genes include the kappa and lambda light chainconstant region genes, the alpha, gamma, delta, epsilon and mu heavychain constant region genes, and the myriad immunoglobulin variableregion genes. Antibodies exist, e.g., as intact immunoglobulins or as anumber of well-characterized fragments produced by digestion withvarious peptidases. This includes, e.g., Fab′ and F(ab)′₂ fragments.Antibodies includes polyclonal and monoclonal antibodies, chimeric,single chain, and humanized antibodies, as well as Fab fragments,including the products of an Fab or other immunoglobulin expressionlibrary.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, polyclonal antibodiesraised to the IαIp complex from a specific species such as rat, mouse,or human can be selected to obtain only those polyclonal antibodies thatare specifically immunoreactive with IαIp complex and not with otherproteins, except for polymorphic variants and alleles of the IαIpcomplex. This selection may be achieved by subtracting out antibodiesthat cross-react with the IαIp complex molecules from other species. Avariety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select antibodiesspecifically immunoreactive with a protein (see, e.g., Harlow & Lane,Antibodies, A Laboratory Manual (1988), for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity). Typically a specific or selective reaction will be atleast twice background signal or noise and more typically more than 10to 100 times background.

“Functionally equivalent” as used herein refers to any protein capableof exhibiting a substantially similar in vivo or in vitro activity asthe IαIp proteins described herein, e.g., effecting a decrease insepsis.

IαIp product structure and purity may be confirmed by, for example, HPLCor other chromatographic method known to one of skill in the art.

As used herein, “IαIp complex” is intended to encompass all naturallyoccurring biologically active variants of the IαIp proteins, includingproteins containing deletions, insertions, additions, and substitutions.A “natural variant” of an IαIp protein is defined as a peptide obtainedfrom plasma having a sequence that is altered by one or more aminoacids. The variant may have “conservative” changes, wherein asubstituted amino acid has similar structural or chemical properties,e.g., replacement of leucine with isoleucine. In other embodiments, avariant may have “nonconservative” changes, e.g., replacement of aglycine with a tryptophan. Similar variations may also include aminoacid deletions or insertions, or both. Guidance in determining which andhow many amino acid residues may be substituted, inserted or deletedwithout abolishing biological or immunological activity may be foundusing computer programs well known in the art, for example, DNASTARsoftware.

A “deletion” is defined as a change in either amino acid or nucleotidesequence in which one or more amino acid or nucleotide residues,respectively, are absent.

An “insertion” or “addition” is that change in an amino acid ornucleotide sequence which has resulted in the addition of one or moreamino acid or nucleotide residues, respectively, as compared to thenaturally occurring IαIp complex.

A “substitution” results from the replacement of one or more amino acidsor nucleotides by different amino acids or nucleotides, respectively.

The term “biologically active” refers to having structural, regulatoryor biochemical functions of a naturally occurring IαIp complex.Likewise, “immunologically active” defines the capability of thenatural, recombinant or synthetic IαIp complex, or any oligopeptidethereof, to induce a specific immune response in appropriate animals orcells and to bind with specific antibodies.

The term “derivative” as used herein refers to the chemical modificationof a nucleic acid encoding IαIp complex or the encoded IαIp complex.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics ofnatural IαIp complex.

“Purifying,” as used herein, refers to steps or processes of removingunwanted or contaminating proteins or components from IαIp to produce apurified IαIp complex. For example, a plasma fraction containing IαI andPαI in physiological proportion may be run through a series ofchromatography steps to purify the IαIp composition.

“Isolating,” as used herein refers to producing a plasma fraction fromblood plasma, which contains IαI and PαI in physiological proportions.For example, isolating a plasma fraction may be achieved in accordancewith the invention by chromatographing blood plasma. Isolated refers tomaterial removed from its original environment (e.g., the naturalenvironment if it is naturally occurring), and thus is altered “by thehand of man” from its natural state. For example, an isolatedpolypeptide or protein could be a component of blood plasma, or could becontained within a cell and be considered “isolated” because that bloodplasma or particular cell may not be the original environment of thepolypeptide.

Chromatographing, as used herein, may include anion-exchangechromatography. Anion-exchange chromatography may be particle-based, forexample, DEAL Sepharose, DEAE Sephadex A50, Toyopearl DEAE, TMAEFractogel, DEAE Fractogel, or Q-Sepharose. Anion-exchange chromatographymay also be by monolithic support, for example, CIM with immobilizedanion-exchange ligands such as DEAE-CIM or Q-CIM. SEPHAROSE is a tradename of Pharmacia, Inc. of New Jersey for a high molecular weightsubstance for the separation by gel filtration of macromolecules. Anionexchange columns have two components, a matrix and a ligand. The matrixcan be, for example, cellulose, dextrans, agarose or polystyrene. Theligand can be diethylaminoethyl (DEAE), polyethyleneimine (PEI) or aquaternary ammonium functional group. The strength of an anion exchangecolumn refers to the state of ionization of the ligand. Strong anionicexchange columns, such as, those, having a quaternary ammonium ligand,bear a permanent positive charge over a wide pH range. In weak anionexchange columns, such as DEAE and PEI, the existence of the positivecharge depends on the pH of the column. Strong anion exchange columnssuch as Q Sepharose FF, or metal-chelating Sepharose (e.g.,Cu2+-chelating Sepharose) are preferred. Anion exchange columns aregenerally loaded with a low-salt buffer at a pH above the pI ofa-glucosidase.

The IαIp compositions of the invention are preferably from between about85% to about 100% pure. As used herein, the term “pure” refers to theIαIp composition that is removed from its natural environment, isolatedor separated, and is at least between about 85% to about 100% free,preferably 90% free, and more preferably 95% free from other componentswith which it is naturally associated. In preferred embodiments, asubstantially purified protein will constitute more than 85%, 87.5%,90%, 92.5%, 95%, 99% or even more of the proteins in the composition.

A peptide, polypeptide or protein that is “purified to homogeneity,” asapplied to the present invention, means that the peptide, polypeptide orprotein has a level of purity where the peptide, polypeptide or proteinis substantially free from other proteins and biological components. Anysuitable materials and methods can be used to perform the isolation stepor steps of blood plasma to obtain purified IαIp.

Typically, preparation of IαIp involves isolation of the sample andcollection of fractions determined to contain the proteins of interest.Methods of isolation include, for example, solid phase extraction,chromatography, for example anion-exchange chromatography, sizeexclusion chromatography, ion exchange chromatography, heparinchromatography, affinity chromatography, sequential extraction, gelelectrophoresis and liquid chromatography. Preparation may also includepurifying, which may include the steps of chromatography, for example,ion exchange chromatography, heparin chromatography, affinitychromatography, sequential extraction, gel electrophoresis and liquidchromatography.

In one embodiment of the invention, a sample can be purified twice byanion exchange chromatography. Anion exchange chromatography allowspurification of the proteins in a sample roughly according to theircharge characteristics. For example, a Q anion-exchange resin can beused (e.g., Q HyperD F, Biosepra), and a sample can be sequentiallyeluted with eluants having different pH's. Anion exchange chromatographyallows separation of biomolecules in a sample that are more negativelycharged from other types of biomolecules. Proteins that are eluted withan eluant having a high pH is likely to be weakly negatively charged,and a fraction that is eluted with an eluant having a low pH is likelyto be strongly negatively charged. Thus, in addition to reducingcomplexity of a sample, anion exchange chromatography separates proteinsaccording to their binding characteristics.

In yet another embodiment, a sample can be further purified by heparinchromatography. Heparin chromatography allows further purification ofthe IαIp complexes in a sample also on the basis of affinity interactionwith heparin and charge characteristics. Heparin, a sulfatedmucopolysaccharide, will bind IαIp complexes with positively chargedmoieties and a sample can be sequentially eluted with eluants havingdifferent pH's or salt concentrations. IαIp complexes eluted with aneluant having a low pH are more likely to be weakly positively charged.IαIp complexes eluted with an eluant having a high pH are more likely tobe strongly positively charged. Thus, heparin chromatography alsoreduces the complexity of a sample and separates IαIp complexesaccording to their binding characteristics.

IαIp complexes may be may be captured with capture reagents immobilizedto a support, such as any biochip, a multiwell microtiter plate, aresin, or nitrocellulose membranes that are subsequently probed for thepresence of proteins. In particular, the IαIp complexes of thisinvention may be captured on Surface-Enhanced LaserDesorption/Ionization (SELDI) protein biochips. Capture can be on achromatographic surface or a biospecific surface. Any of the SELDIprotein biochips comprising reactive surfaces can be used to capture anddetect the IαIp complexes of this invention. These biochips can bederivatized with the antibodies that specifically capture the IαIpcomplexes, or they can be derivatized with capture reagents, such asprotein A or protein G that bind immunoglobulins. Then the IαIpcomplexes can be captured in solution using specific antibodies and thecaptured IαIp complexes isolated on chip through the capture reagent.

Various methods for quantifying the degree of purification of proteins,polypeptides, or peptides will be known to those of skill in the art inlight of the present disclosure. These include, for example, determiningthe specific protein activity of a fraction, or assessing the number ofpolypeptides within a fraction by gel electrophoresis.

In addition to those techniques described in detail herein below,various other techniques suitable for use in protein purification willbe well known to those of skill in the art. These include, for example,precipitation with ammonium sulfate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite, lectinaffinity, immunoaffinity chromatography and other affinitychromatography steps; isoelectric focusing; gel electrophoresis, HPLC;and combinations of such and other techniques. Furthermore, if viewed asdesirable, additional purification steps can be employed usingapproaches that are standard in this art. These approaches are fullyable to deliver a highly pure preparation of the protein.

In other embodiments, gel chromatography, or molecular sievechromatography may be used. Gel chromatography is a special type ofpartition chromatography that is based on molecular size. The theorybehind gel chromatography is that the column, which is prepared withtiny particles of an inert substance that contain small pores, separateslarger molecules from smaller molecules as they pass through or aroundthe pores, depending on their size. As long as the material of which theparticles are made does not adsorb the molecules, the sole factordetermining rate of flow is the size. Hence, molecules are eluted fromthe column in decreasing size, so long as the shape is relativelyconstant. Gel chromatography is unsurpassed for separating molecules ofdifferent size because separation is independent of all other factorssuch as pH, ionic strength, temperature, etc. There also is virtually noadsorption, less zone spreading and the elution volume is related in asimple matter to molecular weight.

In other embodiments, affinity chromatography may be used. Affinitychromatography is a chromatographic procedure that relies on thespecific affinity between a substance to be isolated and a molecule thatit can specifically bind to. This is, for example, a receptor-ligandtype interaction. The column material may be synthesized by covalentlycoupling one of the binding partners to an insoluble matrix. The columnmaterial may then be able to specifically adsorb the substance from thesolution. Elution occurs, for example, by changing the conditions tothose in which binding will not occur (e.g., alter pH, ionic strength,and temperature.).

The methods described herein are amenable for large-scale production,and result in proteins including, the IαIp complex and H4 in a formsuitable for therapeutic administration.

Any of the isolation or purification steps of the processes describedherein may be repeated to obtain a higher degree of purity, if desired.The chromatography steps may be in batch or in column format.

Preferred processes for producing a blood plasma-derived IαIpcomposition of the invention include isolating from blood plasma aplasma fraction containing IαI and PαI, wherein the IαI and PαI arepresent in a physiological proportion; and purifying the plasma fractionto obtain an IαIp composition with a purity of from between about 85% toabout 100% pure.

Processes according to the invention may also include furtherpurification of the plasma fraction, for example, by passing to aheparin affinity column and collecting the flow through (unbound)fraction.

Processes useful in practicing the invention may also include virusinactivating the plasma fraction and/or the purified IαIp before and/orafter the purification and isolation steps. Typical processes includevirus inactivating by a solvent/detergent treatment or thermalinactivation. Thermal in activations, or pasteurization of thecompositions of the invention may be for example at a temperature ofbetween about 55° to about 65° C. or dry heat at 70 to 120° C.

For solvent detergent treatments, a specific combination of solvent anddetergents, such as, 0.3% tri-n-butylphosphate (TnBP) combined with 1%Tween-80, at 24° C. for 6 hours, is effective to inactivate envelopedviruses (Horowitz et al (1985) Transfusion 25, pp. 516-522).Alternatively, pasteurization of the fraction or purified IαIp at 55-65°C. in the presence of stabilizers may be sufficient to inactivate anyviruses present.

During the purification or isolation step(s) stabilizers may be added.The final composition of IαIp may also contain stabilizers. For example,suitable stabilizers include albumin, polyethylene glycol, alpha,alpha-trehalose, amino acids, salts, glycerol, omega-amino acids such aslysine, polylysine, arginine, epsilon amino caproic acid and tranexamicacid, sugars, such as sucrose, or combinations thereof.

The IαIp proteins and compositions of the invention may be used to treata human disease. Such diseases include, for example, acute inflammatorydiseases, sepsis, severe shock, septic shock, rheumatoid arthritis,cancer, cancer metastasis, preterm labor and infectious diseases. TheIαIp compositions used to treat disease may be made by isolating fromblood plasma a plasma fraction containing IαI and PαI, wherein the IαIand PαI are present in a physiological proportion; and purifying theplasma fraction to obtain an IαIp composition with a purity of IαIpranging from about 85% to about 100% pure.

The invention also encompasses pharmaceutical compositions of IαIp.Pharmaceutical compositions of IαIp may be any of the IαIp compositionsdescribed herein in a therapeutically effective amount with apharmaceutically acceptable carrier.

For example, a pharmaceutical composition according to the invention maybe a therapeutically effective amount of an IαIp composition which is amixture of inter-alpha inhibitor protein (IαI) and pre-alpha protein(PαI), wherein the IαI and the PαI are present in said mixture in aphysiological proportion ranging from about 85% to about 100% pure. Apharmaceutical composition may also be a therapeutically effectiveamount of an IαIp composition which is a mixture of inter-alphainhibitor protein (IαI) and pre-alpha protein (PαI), wherein the IαI andthe PαI are present in said mixture in a physiological proportioncomprising a light chain of inter-alpha inhibitor protein associatedwith at least one of three heavy chains H1, H2 and H3 or H1, H2, H3 andH4. A pharmaceutical composition may also be a therapeutically effectiveamount of an IαIp composition which is a mixture of inter-alphainhibitor protein (IαI) and pre-alpha protein (PαI), wherein the IαI andthe PαI are present in said mixture in a physiological proportion andhaving a high trypsin inhibitory specific activity. A pharmaceuticalcomposition may also be a therapeutically effective amount of an IαIpcomposition which is a mixture of inter-alpha inhibitor protein (IαI)and pre-alpha protein (Pa), wherein the IαI and the PαI are present insaid mixture in a physiological proportion and having a half life ofgreater than one hour, five hours, or ten hours.

The term “pharmaceutically acceptable carrier or adjuvant” refers to acarrier or adjuvant that may be administered to a subject, together witha composition of this invention, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the composition.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying drug delivery systems (SEDDS) such asd-α-tocopherol polyethyleneglycol 1000 succinate, surfactants used inpharmaceutical dosage forms such as Tweens or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, orchemically modified derivatives such as hydroxyalkylcyclodextrins,including 2- and 3-hydroxypropyl-β-cyclodextrins, or other solubilizedderivatives may also be advantageously used to enhance delivery ofcompositions described herein.

The pharmaceutical compositions of this invention may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir, preferably by oraladministration or administration by injection.

The pharmaceutical compositions of this invention may contain anyconventional non-toxic pharmaceutically-acceptable carriers, adjuvantsor vehicles. In some cases, the pH of the formulation may be adjustedwith pharmaceutically acceptable acids, bases or buffers to enhance thestability of the formulated composition or its delivery form. The termparenteral as used herein includes subcutaneous, intracutaneous,intravenous, intramuscular, intraarticular, intraarterial,intrasynovial, intrasternal, intrathecal, intralesional and intracranialinjection or infusion techniques. Suitable methods of administration maybe as a tablet, capsule, or by intravenous injection. Injectable formsof administration are particularly preferred.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, or carboxymethyl cellulose or similar dispersing agentswhich are commonly used in the formulation of pharmaceuticallyacceptable dosage forms such as emulsions and or suspensions. Othercommonly used surfactants such as Tweens or Spans and/or other similaremulsifying agents or bioavailability enhancers which are commonly usedin the manufacture of pharmaceutically acceptable solid, liquid, orother dosage forms may also be used for the purposes of formulation.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, emulsions and aqueous suspensions,dispersions and solutions. In the case of tablets for oral use, carrierswhich are commonly used include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions and/or emulsions areadministered orally, the active ingredient may be suspended or dissolvedin an oily phase is combined with emulsifying and/or suspending agents.If desired, certain sweetening and/or flavoring and/or coloring agentsmay be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a composition of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

Topical administration of the pharmaceutical compositions of thisinvention is useful when the desired treatment involves areas or organsreadily accessible by topical application. For application topically tothe skin, the pharmaceutical composition should be formulated with asuitable ointment containing the active components suspended ordissolved in a carrier. Carriers for topical administration of thecompositions of this invention include, but are not limited to, mineraloil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene composition, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active composition suspended ordissolved in a carrier with suitable emulsifying agents. Suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water. The pharmaceuticalcompositions of this invention may also be topically applied to thelower intestinal tract by rectal suppository formulation or in asuitable enema formulation. Topically-transdermal patches are alsoincluded in this invention.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

Methods of treating an inflammation related disorders, for example,rheumatoid arthritis, septic and septic shock, head trauma/injury andmeningitis, inflammatory bowel diseases (Crohn's Disease), chronicobstructive pulmonary disease, rhinitis; cancer, preterm labor, or aninfectious disease in a subject according to the invention may includeadministering a therapeutically effective amount of an IαIp compositionthat is produced according to the methods of the invention.

Compositions herein are administered in a dosage ranging from about 1 to50 mg/kg of body weight, preferably dosages between 500 mg and 1000mg/dose, every 4 to 120 hours, or according to the requirements of theparticular drug. The methods herein contemplate administration of aneffective amount of composition to achieve the desired or stated effect.Typically, the pharmaceutical compositions of this invention will beadministered from about 1 to about 6 times per day or alternatively, asa continuous infusion. Such administration can be used as a chronic oracute therapy. The amount of active ingredient that may be combined withthe carrier materials to produce a single dosage form will varydepending upon the host treated and the particular mode ofadministration. A typical preparation will contain from about 5% toabout 95% active composition (w/w). Alternatively, such preparationscontain from about 20% to about 80% active composition.

Lower or higher doses than those recited above may be advantageous.Specific dosage and treatment regimens for any particular subject willdepend upon a variety of factors, including the activity of the specificcomposition employed, the age, body weight, general health status, sex,diet, time of administration, rate of excretion, drug combination, theseverity and course of the disease, condition or symptoms, the subject'sdisposition to the disease, condition or symptoms, and the judgment ofthe treating physician.

Upon improvement of a condition, a maintenance dose of a composition orcombination of this invention may be administered, if necessary.Subsequently, the dosage or frequency of administration, or both, may bereduced, as a function of the symptoms, to a level at which the improvedcondition is retained when the symptoms have been alleviated to thedesired level, treatment should cease. Subjects may, however, requireintermittent treatment on a long-term basis upon any recurrence ofdisease symptoms. Improvement of the condition may also be judged basedupon the level of IαIp in the liver. If normal physiological levels ofIαIp are found in the liver and the patients symptoms as judged by thesubject or the treating physician are judged to be improved, the subjectmay treated with a maintenance dose.

When the compositions of this invention comprise a combination of anIαIp composition and one or more additional therapeutic or prophylacticagents, both the composition and the additional agent should be presentat dosage levels of between about 1 to 100%, and more preferably betweenabout 5 to 95% of the dosage normally administered in a monotherapyregimen. The additional agents may be administered separately, as partof a multiple dose regimen, from the compositions of this invention.Alternatively, those agents may be part of a single dosage form, mixedtogether with the compositions of this invention in a singlecomposition.

Methods for treating a subject for acute inflammatory disease, sepsis,severe shock, septic shock, rheumatoid arthritis, cancer, cancermetastasis, infectious disease, and/or preterm labor, include the stepsof determining the pre-treatment level of one or more of IαI, PαI, IαIp,H3, H4, H1, H2, and LC; and administering a therapeutically effectiveamount of IαIp to the subject. Pre-treatment levels of IαI, PαI, IαIp,H3, H4, H1, H2, and LC are the levels of the proteins in the subjectprior to the first administration of IαIp or any of the IαIp complexproteins. Post-treatment levels are the levels of IαIp measured afteradministration of IαIp. The methods of the invention include determiningthe post-treatment levels of one or more of IαI, PαI, IαIp, H3, H4, H1,H2, and LC after an initial period of treatment with IαIp. A modulationin the level of IαIp is an indication that the treatment is producing afavorable clinical response. The initial period of treatment may be thetime required to achieve a steady-state plasma concentration of theIαIp.

The level of IαI, PαI, IαIp, H3, H4, H1, H2, and/or LC may bedetermined, for example, by immunological methods. For example, IαIpcomplexes can be detected and/or measured by a variety of detectionmethods including for example, gas phase ion spectrometry methods,optical methods, electrochemical methods, atomic force microscopy, radiofrequency methods, surface plasmon resonance, ellipsometryimmunological, and atomic force microscopy methods.

In another embodiment, an immunoassay can be used to detect and analyzeIαIp complexes in a sample. This method comprises: (a) providing anantibody that specifically binds to an IαIp complex; (b) contacting asample with the antibody; and (c) detecting the presence of a complex ofthe antibody bound to the IαIp complex in the sample. Suitableantibodies for use in the methods of the invention include, MAb 69.31,MAb 69.26, anti-IαIp polyclonal antibody (R16 or R20), and anti-bikuninmonoclonal or polyclonal antibody.

An immunoassay is an assay that uses an antibody to specifically bind anantigen (e.g., an IαIp complex). The immunoassay is characterized by theuse of specific binding properties of a particular antibody to isolate,target, and/or quantify the antigen. The phrase “specifically (orselectively) binds” to an antibody or “specifically (or selectively)immunoreactive with,” when referring to a protein or peptide, refers toa binding reaction that is determinative of the presence of the proteinin a heterogeneous population of proteins and other biologics. Thus,under designated immunoassay conditions, the specified antibodies bindto a particular protein at least two times the background and do notsubstantially bind in a significant amount to other proteins present inthe sample. Specific binding to an antibody under such conditions mayrequire an antibody that is selected for its specificity for aparticular protein. For example, polyclonal antibodies raised to an IαIpcomplex from specific species such as rat, mouse, or human can beselected to obtain only those polyclonal antibodies that arespecifically immunoreactive with that IαIp complex and not with otherproteins, except for polymorphic variants and alleles of the IαIpcomplex. This selection may be achieved by subtracting out antibodiesthat cross-react with the IαIp complex molecules from other species.

Subjects suitable for treatment with IαIp may be identified as havinginflammation, trauma/injury, tumor invasion, tumor metastasis, sepsis,septic shock, or an infectious disease. The subject may beself-identified or diagnosed by a medical practitioner as havinginflammation, tumor invasion, tumor metastasis, sepsis, septic shock, oran infectious disease. The subjects may be primates, humans, or otheranimals.

Methods also include the co-administration of other therapeutic agents.For example, the additional therapeutic agents may be anticancer agents,anti-inflammatory agents, anti-coagulants or immunomodulators. Forexample, dideoxynucleosides, e.g. zidovudine (AZT), 2′,3′-dideoxyinosine(ddI) and 2′,3′-dideoxycytidine (ddC), lamivudine (3TC), stavudine(d4T), and TRIZIVIR (abacavir+zidovudine+lamivudine), nonnucleosides,e.g., efavirenz (DMP-266, DuPont Pharmaceuticals/Bristol Myers Squibb),nevirapine (Boehringer Ingleheim), and delaviridine (Pharmacia-Upjohn),TAT antagonists such as Ro 3-3335 and Ro 24-7429, protease inhibitors,e.g., indinavir (Merck), ritonavir (Abbott), saquinavir (HoffmannLaRoche), nelfinavir (Agouron Pharmaceuticals), 141 W94(Glaxo-Wellcome), atazanavir (Bristol Myers Squibb), amprenavir(GlaxoSmithKline), fosamprenavir (GlaxoSmithKline), tipranavir(Boehringer Ingleheim), KALETRA (lopinavir+ritonavir, Abbott), and otheragents such as 9-(2-hydroxyethoxymethyl)guanine (acyclovir), interferon,e.g., alpha-interferon, interleukin II, and phosphonoformate(Foscarnet), or entry inhibitors, e.g., T20 (enfuvirtide,Roche/Trimeris) or UK-427,857 (Pfizer), levamisol or thymosin,cisplatin, carboplatin, docetaxel, paclitaxel, fluorouracil,capecitabine, gemcitabine, irinotecan, topotecan, etoposide, mitomycin,gefitinib, vincristine, vinblastine, doxorubicin, cyclophosphamide,celecoxib, rofecoxib, valdecoxib, ibuprofen, naproxen, ketoprofen,dexamethasone, prednisone, prednisolone, hydrocortisone, acetaminophen,misonidazole, amifostine, tamsulosin, phenazopyridine, ondansetron,granisetron, alosetron, palonosetron, promethazine, prochlorperazine,trimethobenzamide, aprepitant, diphenoxylate with atropine, and/orloperamide. Anti-coagulants such as Anti-thrombin III, activated ProteinC and protease inhibitors such as furin inhibitors

Methods also include predicting a response to an IαIp therapy byassaying a sample obtained from a subject to detect the level of one ormore of IαI, PαI, IαIp, H3, H4, H1, H2, and LC; wherein the detectedlevels identifies a subject that may respond favorably to IαIp therapy.For example, a decrease in a detectable level of IαI and/or PαIindicates that a subject may benefit from the administration of IαIp.

Methods also include monitoring the progress of a subject being treatedwith an IαIp therapy by determining the pre-treatment level of one ormore of IαI, PαI, IαIp, H3, H4, H1, H2, and LC; administering atherapeutically effective amount of IαIp to the subject; and determiningthe level of one or more of the levels in the subject after an initialperiod of treatment with the IαIp, wherein an increase of the level inthe subject following treatment with IαIp indicates that the subject islikely to have a favorable clinical response to treatment with IαIp.

Kits for IαIp therapy may include one or more of IαI, PαI, IαIp, H3, H4,H1, H2, and LC; and instructions for therapeutic use. Kits are alsocontemplated that have the IαIp compositions described herein andinstructions for use. For example, a kit may have IαI, PαI, andinstructions that provide information regarding dosage, form ofadministration, and storage conditions.

A container including IαIp and a label or package insert withinstructions for administering the IαIp to a subject are alsocontemplated. The instructions may provide instructions for dosage, formof administration, and storage conditions.

EXAMPLES

It should be appreciated that the invention should not be construed tobe limited to the examples which are now described; rather, theinvention should be construed to include any and all applicationsprovided herein and all equivalent variations within the skill of theordinary artisan.

Example 1 Characterization of H4 as a Part of the IαIp Complex

SELDI-TOF mass spec analysis indicated that heavy chain 4 (H4) is alsopresent in both 125 kDa band (PαI) and 250 kDa band (IαI) (data notshown). The presence of H4 in the 250 kDa band is more pronounced. Thissuggests that another complex protein other than IαI (H1+H2+LC) and PαI(H3+LC) might be present in some compositions of the invention. So far,H4 has not been described in the complexed form. Free H4 is smaller than125 or 250 kDa. Due to the mass spec results, we think that H4 might bein complex with bikunin (the light chain) or something else.

Example 2 Animal Model of Sepsis

Male Sprague-Dawley rats (275-325 g) were housed in atemperature-controlled room on a 12-h light/dark cycle and fed astandard Purina rat chow diet. Prior to the induction of sepsis, ratswere fasted overnight but allowed water ad libitum. Rats wereanesthetized with isoflurane inhalation and the ventral neck, abdomenand groin were shaved and washed with 10% povidone iodine. A 2-cmmidline abdominal incision was performed. The cecum was exposed, ligatedjust distal to the ileocecal valve to avoid intestinal obstruction,punctured twice with an 18-gauge needle, squeezed slightly to allow asmall amount of fecal matter to flow from the holes, and returned to theabdominal cavity; the abdominal incision was then closed in layers.Sham-operated animals (i.e., control animals) underwent the sameprocedure with the exception that the cecum was neither ligated norpunctured. The animals were resuscitated with 3 ml/100 g BW normalsaline subcutaneously immediately after surgery. Animals were thenanesthetized at various intervals after cecal ligation and puncture(CLP) or sham operation for collection of tissue samples. Allexperiments were performed in accordance with the National Institutes ofHealth guidelines for the use of experimental animals. This project wasapproved by the Institutional Animal Care and Use Committee of the NorthShore-Long Island Jewish Research Institute.

Preparation and Administration of Human IαIp

Human IαIp (both IαI and PαI) was isolated as a by-product of aprocedure designed for purifying coagulation factor VIII from humanplasma. The procedure involves ion exchange and size-exclusionchromatography of cryoprecipitates. A purity of approximately 70% wasachieved after the chromatographic separation. This IαIp containingpreparation has few side effects in terms of toxicity, thrombogenicity,or hypotension. At 1, 5, 10 or 10 and 20 h after the onset of sepsis,the left femoral vein was cannulated with a polyethylene-50 tubing underisoflurane anesthesia Human IαIp concentrates at a dose of 30 mg/kg BWor an equivalent volume of vehicle (normal saline, 1.5 ml/rat) wereadministered intravenously via the femoral catheter over 30 min at aconstant infusion rate. Administration of human IαIp, in previousexperiments, did not alter mean arterial pressure or heart rate duringthe period of infusion or thereafter (data not shown).

RNA Extraction and Determination of Hepatic Bikunin Genes

The liver may be the major source of bikunin. Therefore, we measuredbikunin mRNA expression in the liver. Total RNA was extracted from theliver by Tri-Reagent (Molecular Research Center, Cincinnati, Ohio). Onehundred mg of liver tissue was homogenized in 1.5 ml Tri-Reagent,separated into aqueous and organic phases by chloroform addition, andcentrifuged. RNA was precipitated from the aqueous phase by addition ofisopropanol, and washed with ethanol. The pellet was dissolved in 0.1%DEPC-treated, deionized distilled water. RNA concentration and puritywere determined by measuring the absorbance at 260 and 280 nm. Five μgof RNA from each tissue was reverse-transcribed in a 20 μl reactionvolume containing 50 mM KCl, 10 mM Tris-HCl, 5 mM MgCl2 MgCl₂, 1 mMdNTP, 20 U RNase inhibitor, 2.5 mM oligo d(T) 16 primer and 50 U reversetranscriptase. The reverse transcription reaction solution was incubatedat 42° C. for 1 h, followed by heating at 95° C. for 5 min. One μl cDNAwas amplified with 0.15 μM each of 3′ and 5′ primers, specific for ratbikunin (633 bp) (5′ TGA CGA ATA TGC CAT TTT CC 3′ (SEQ ID NO: 2), 5′CCA5′ CCA CAG TAC TCC TTG CAC TCC 3′ (SEQ ID NO: 3)) (accession No.S87544), rat glyceraldehydes-3-phosphate-dehydrogenase 7 (G3PDH) (24)(983 bp) (5′ TGA AGG TCG GTG TCA ACG GAT TTG GC 3′ (SEQ ID NO: 4), 5′CAT5′ CAT GTA GGC CAT GAG GTC CAC CAC 3′ (SEQ ID NO: 5)) in 25 μl of PCRmixture containing 50 mM KCl, 10 mM Tris-HCl, 2 mM MgCl2 MgCl₂, 0.2 mMdNTP and 0.7 U AmpliTaq DNA polymerase. PCR was carried out in a Bio-Radthermal cycler. Following RT-PCR, 5 μl of the reaction mixture waselectrophoresed in 1.2% TBE-agarose gel containing 0.22 μg/ml ethidiumbromide. The gel was then developed and band intensities were normalizedby G3PDH using the Bio-Rad image system (Hercules, Calif.).

Radioiodination of Protein and Determination of IαIp Half-Life

Purified IαIp was radioiodinated with Na¹²⁵I (Amersham, ArlingtonHeights, Ill.) using 1,3,4,6-tetrachloro-3a-6a-diphenyl glycoluril(IODO-GEN iodination reagent; Pierce, Rockford, Ill.). Theunincorporated ¹²⁵I was removed by applying the reaction mixture to anExcellulose GF-5 desalting column (Pierce). Radioactivity was determinedin a gamma counter (Pharmacia-LKB, Piscataway, N.J.). At 12 h after CLPor sham operation, the animals were anesthetized with isofluraneinhalation. A steady state of sedation was maintained with a subsequentintravenous injection of sodium pentobarbital (˜30 mg/kg BW).Polyethylene-50 catheters were placed in the right jugular vein and leftfemoral artery, and a bolus injection of ¹²⁵I-labeled IαIp (˜500,000cpm/rat) was administered through the jugular cannula. The remainingradioactivity in the syringe was measured with a gamma counter, and theradioactivity counts were subtracted from the initial preinjectioncounts to determine the net injected radioactivity. Blood samples werecollected immediately after the injection and then every 2 h for aperiod of 8 h for determining the half-life (t_(1/2)) of ¹²⁵I-IαIp inthe circulation. The radioactivity (cpm) in each sample was measuredwith a gamma counter. The t_(1/2) was calculated according to Wu R, ZhouM, Cui X, et al: Ghrelin clearance is reduced at the late stage ofpolymicrobial sepsis. Int J Mol Med. 2003; 12:777-782.

Survival Study

CLP was performed as described above. At 1, 5, 10 or 10 and 20 h afterCLP, human IαIp concentrate (30 mg/kg BW) or vehicle (normal saline, 1.5ml/rat, at 1 h after CLP or 10 and 20 h after CLP) was infusedintravenously. At 20 h after CLP, the necrotic cecum was excised and theabdominal cavity was washed twice by using 40 ml of warm, sterilizednormal saline solution. The abdominal incision then was closed inlayers. The procedure of cecal excision in CLP animals was performed tomimic the clinical situation in which septic focus should be removedwhenever possible. The animals then were allowed food and water adlibitum and were monitored for 10 days to record survival.

Statistical Analysis

Results are expressed as means ±SE. One-way analysis of variance (ANOVA)and Tukey's test were used to compare different groups of experimentalanimals. The survival rate was estimated by Kaplan-Meier method andcompared by the log-rank test. Differences in values were consideredsignificant if P<0.05. 9

Alterations in Bikunin mRNA Expression after CLP

Bikunin is an active part of IαIp. The liver is the major source ofbikunin. Therefore, we chose bikunin mRNA expression in the liver toreflect the production of IαIp. As shown in FIG. 3, mRNA expression ofbikunin in the liver did not change at 5 h after CLP, however, a 32%decrease was found at 20 h after CLP as compared with sham operatedanimals (P<0.05).

Alterations in t_(1/2) of ¹²⁵I-IαIp after CLP

Half-life of IαIp was estimated by measuring the changes of blood levelsof radioactive labeled IαIp injected at 12 h after the onset of sepsis.As indicated in FIG. 4, the t_(1/2) of 125I-IαIp was significantlyincreased from 5.6±0.3 h to 11.8±2.7 h (P<0.05) after CLP.

Effects of IαIp on Survival Rate

The survival rate after CLP and cecal excision with single time vehicleadministration (at 1 h after CLP) was 75% at day 2 and decreased to 50%at days 5-10 FIG. 5). Administration of human IαIp at 1 h after CLP,however, improved the survival rate to 92% throughout the 10-dayobservation period (P<0.05; FIG. 5). Although administration of humanIαIp at 5 or 10 h after CLP improved the survival rate to 64% and 73%respectively, these improvements were not statistically significant(FIG. 6). The survival rate after CLP and cecal excision with two timesvehicle administration (at 10 and 20 h after CLP) was 56% at day 2 anddecreased to 44% at days 5-10 (FIG. 7), which was not significantlydifferent as compared with one time vehicle administration (FIG. 5).Administration of human IαIp at 10 and 20 h after CLP, however, improvedthe survival rate to 81% throughout the 10-day observation period, whichwas significantly different as compared with vehicle group (P<0.05; FIG.7).

Sepsis is a clinical syndrome characterized by systemic inflammation,coagulopathy, respiratory failure, myocardial dysfunction, renalinsufficiency, and neurocognitive defects. It is generally assumed thatthis syndrome results from an excessive triggering of endogenousinflammatory mediators by the invading microorganisms. These mediatorsinclude substances released by activated monocytes, macrophages,endothelial cells and neutrophils such as cytokines, reactive oxygenspecies and proteases. In severe inflammatory response, various bloodand tissue cells, including polymorphonuclear granulocytes, releaselysosomal proteinases extracellularly and into the circulation. Suchproteases as well as normally intracellular oxidizing agents producedduring phagocytosis, can trigger tissue and organ damage and enhance thenonspecific proteolysis of plasma clotting and complement factors. Therelease of neutrophil proteinases, especially human leukocyte elastase,has been implicated in the progress of complications in subjects withsepsis. Their plasma levels are in close correlation with the severityof infection-induced inflammation and highly predictive of forthcomingorgan failure.

During septic shock in humans, in addition to elevated activities ofproteases, decreased plasma levels of IαIp have been reported. Subjectswith severely decreased concentrations of IαIp have a higher mortalityrate. Our results indicate that the gene expression of bikunin in theliver is significantly lower in the CLP animals than in thesham-operated animals. The expression of mRNA related to proteins in theinter-alpha-inhibitor family has been examined in various tissues inprimates, pigs, and rodents. These studies indicate the genes which arethe source of all members of the IαIp family are primarily transcribedin the liver. Our results also show the gene expression of bikunin inother organs (i.e., intestine and kidneys) was not significantly alteredat 5 or 20 h after CLP as compared to the shams (data not shown).

The significant downregulation observed only in the liver at 20 h afterCLP suggests that this organ might be an important source of IαIp andfurthermore, in late stages of sepsis, bikunin gene expression in theliver is significantly decreased. Therapeutically, bikunin has beenreported to have beneficial effects in humans as a prophylactictreatment to prevent pancreatitis after gastrectomy or to attenuateorgan injury after cardiac surgery. Studies that examined the effects ofbikunin in an acute canine model of lethal E. coli bacteremia showedsimilar results with an improvement in hemodynamic variables andnormalization of cardiac output and mean arterial pressure. Because theplasma half-life of bikunin is very short (approximately 10 min), itappears important to prolong the half-life of bikunin to maintain thesustained beneficial effects of this agent. Our results show that thehalf-life of IαIp in sham-operated animals is 5.6 h. When we injectedradioactive labeled IαIp at 12 h after the onset of sepsis, we found thehalf-life of IαIp was prolonged to 11.8 h. These results indicate thatIαIp clearance was significantly decreased during sepsis. However, evenwith decreased clearance the plasma levels of IαIp remain significantlylower in septic subjects. Our previous study has shown thatadministration of low purity IαIp early after the onset of sepsis (i.e.,1 h post-CLP) maintained cardiac output and systemic oxygen delivery andincreased systemic oxygen consumption and systemic oxygen extractionratio. Moreover, IαIp downregulated TNF-a production and attenuatedhepatocellular injury and lactic acidosis at 20 h after CLP. Inaddition, IαIp administration at 1 h after the onset of sepsis improvedsurvival in septic animals.

Human IαIp proteins were isolated as a by-product of industrial scaleplasma fractionation. The isolation method highly, simultaneouslyenriches the major plasma form of bikunin-containing proteins (IαI andPαI). Thus, in this preparation, a physiologic composition of plasmaIαIp is obtained. The results of the mortality study performed indicatethat administration of IαIp at 1 h after CLP improved the survival ratefrom 50% to 92% at 10 days after CLP and cecal excision. Althoughadministration of human IαIp at 5 or 10 h after CLP improved thesurvival rate to 64% and 73% respectively, these improvements were notstatistically significant.

Administration of human IαIp at 10 and 20 h after CLP, however,significantly improved the survival rate from 44% to 81%. Thus, IαIpappear to be a useful adjunct for improving survival during theprogression of polymicrobial sepsis. In summary, bikunin gene expressionin the liver decreased during sepsis and the half-life of IαIp increasedfrom 5.6±0.3 h to 11.8±2.7 h, suggesting downregulation of bikunin insepsis despite a decrease clearance. Administration of IαIp at 1 h afterCLP improved the survival rate from 50% to 92%, whereas there was nosignificant improvement when IαIp was administrated at 5 or 10 h afterCLP. However, double injection of IαIp at 10 and 20 h after CLPincreased the survival rate from 44% to 81%. Delayed but repeatedadministration of human IαIp improve survival after CLP.

Example 3 Purification of IαIp

After application of dialyzed or ultra/diafiltrated eluate aftersolid-phase extraction with DEAF Sephadex A50, weakly bound componentsare eluted from DEAE-Sepharose FF column with 0.005 M sodiumcitrate/0.0055 M sodium phosphate buffer, pH 6.0 containing 0.28 Msodium chloride (described in Hoffer et al., Journal of Chromatography B669 (1995) 187-196). In the previous step, the column was washed with0.005 M sodium citrate/0.0055 M sodium phosphate buffer, pH 6.0,containing 0.20 M sodium chloride.

After dialysis or ultrafiltration/diafiltration (UF/DF) against 0.005 Msodium phosphate buffer pH 7.0, the eluate was applied tohydroxylapatite column. The IαIp proteins do not bind to the column andare collected as flow through fraction. The contaminating proteins,mainly FII, FVII and FX, can be eluted using a gradient with anincreasing concentration of sodium phosphate buffer. The IαI/PαIfraction contains more than 90% of target proteins, mainly IαI and PαI.

Example 4 Purification of IαIp from a Factor IX Flow Through Fraction

Unbound proteins from Heparin Sepharose affinity chromatography (L.Hoffer et al., J. of Chromatography B), are applied to a DEAE-SepharoseFF anion-exchange column. After washing the column with a minimum ofthree column volumes of 0.005 M phosphate buffer, pH 7.0, IαIp/PαIcontaining fractions were eluted with 0.005 M phosphate buffer, pH 7.0,containing 0.55 M sodium chloride (elution buffer). The eluate containsabout 30-40% IαI/PαI. After dialysis or ultrafiltration/diafiltration(UF/DF) against 0.005 M sodium phosphate buffer, pH 7.0, the eluate fromDEAE Sepharose EF was applied to a hydroxylapatite column. The IαI/PαIdo not bind to the column and are collected as a flow-through fraction.The contaminating proteins, mainly FII and FX can be eluted using agradient with increasing concentration of sodium phosphate buffer. Thepurified IαI/PαI fraction contains more than 90% of the target proteins

Example 5

Eluant from a solid-phase extraction of cryopoor plasma on DEAE-SephadexA50 (L. Hoffer et al., J. of Chromatography B) or Q-Sephadex A50 (D.Josic et al., Thrombosis Research, cf. above) was applied to a DEAC-CIMtube monolith with a column volume of 80 mL (cf. K. Branovic et al., Jof Chromatography A, 903 (2000) 21-32). The unbound fraction(flow-through) was collected. The column was subsequently washed withthree column volume of 0.02 M Tris-HCl, pH 7.4 (equilibration buffer).Bound proteins were eluted in first step with 0.02 M Tris-HCl, pH 7.4,containing 0.35 Mol/L sodium chloride (Eluate 1) and in a second stepwith 0.02 M Tris-HCl, pH 7.4, containing 0.55 M sodium chloride (Elution2). IαI/PαI are found in flow-through fractions and Eluate 1.Flow-through fractions contain about 35-45% IαI/PαI. The amount of thesetarget proteins in Eluate 1 is between 20-30%. Fractions containingIαI/PαI were subjected to dialyses or ultrafiltration/diafiltration(UF/DF) against 0.005 M sodium phosphate buffer, pH 7 and applied to ahydroxylapatite column. IαIp do not bind to the column and are collectedas a flow-through fraction. The flow-through fraction contains more than90% of IαI/PαI.

FIGS. 8a and 8b describe exemplary purification schemes of IαIp in aflow diagram as represented by Examples 3-5.

Example 6 Beneficial Effects of Highly Purified IαIp in the AnimalStudies

Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) inmale Sprague-Dawley rats. Animals were fasted overnight before CLP wasperformed but were allowed water ad libitum. At the time of theexperiment, the rats were anesthetized by methylflurane inhalation, anda 2-cm ventral midline incision was performed. The caecum was exposed,ligated just distally to the ileocecal valve to avoid intestinalobstruction, punctured twice with an 18-gauge needle, squeezed gently toforce out a small amount of feces, and then returned to the abdominalcavity. The abdominal incision was closed in layers, and the animalsreceived 30 mL/kg body weight normal saline solution subcutaneouslyimmediately after CLP as a fluid resuscitation. Two groups of rats withn=12 per group were used in this experiment. The treatment groupreceived highly purified IαIp at 10 and 20 hrs after CLP at 30 mg/kgbodyweight. The control group received saline. At 20 hr after CLP, thenecrotic caecum was excised and the abdominal cavity washed twice byusing 40 mL of warm, sterile normal saline solution. The abdominalincision was then closed in layers. The procedure of cecal excision inCLP animals was performed to mimic the clinical situation in which theseptic focus is removed. The experimental animals were allowed food adlibitum and monitored for 10 days to record the time of death for thenon-survivors. A log-rank test was employed for comparison of mortalityrates among different groups of animals and p values were determined byKaplan-Meier method. A significant increase in survival was observed inthe treatment group compared to the saline control group (81.3% of theanimals in the treatment group survived vs. 44% in the control group (pvalue=0.0293)), suggesting that the highly purified IαIp is biologicallyactive and effective in reducing the sepsis-related death in septicanimals. Results are shown in FIG. 9.

TABLE 1 Comparison of the specific inhibitory activity of IαIp ProteinIαIp IαIp Specific Inh. IαIp preparation conc. conc. purity ActivityPurified from [mg/mL] [mg/mL] [%] [TIU/mg IαIp] Cryoprecipitate 7.305.10 69.86% 1412 ± 47.52 Cryopoor plasma 17.30 17.00 98.27% 1409 ± 42.50TIU = Trypsin inhibitory unit; mean ± SD from three independentexperiments.

Specific inhibitory activity of the highly purified IαIp (from cryo-poorplasma) was calculated and compared with IαIp purified from thecryoprecipitate, a side-fraction of fVIII production. The biologicalactivity of IαIp was measured in a trypsin inhibition assay using thechromogenic substrate L-BAPA (N(alpha)-Benzoyl-L-arginine-4-nitroanilidehydrochloride (Fluka Chemicals). This assay is based on the ability ofIαIp to inhibit the hydrolysis of L-BAPA. Inhibition can be monitored bya decrease in the rate of Δ absorbance/minute at 410 nm. Total proteinconcentration was quantitatively measured by the BioRad protein assayand IαIp concentrations were measured by a competitive ELISA using MAb69.31 as described in Lim et al, J. of Infectious Diseases, 2003). Therewas no significant difference in the specific trypsin inhibitoryactivity between IαIp preparations purified from cryo-poor plasma andcryoprecipitate (p value=0.939), suggesting that IαIp in both fractionshad comparable biological activity.

Example 7 Side Fraction from FIX Purification

The “washing fraction” from the chromatographic purification of clottingfactor FIX on DEAF-Sepharose FF (Josic et al. Journal of Chromatography,citated above). This fraction can be eluted with a 0.01-0.1 M sodiumcitrate/0.005-0.1 M sodium phosphate buffer, pH 6.0 containing 0.25 Msodium chloride. The IαI and PαI are the main components in thisfraction. The FIX containing fraction can be eluted from DEAE SepharoseFF column with a 0.01-0.1 M sodium citrate/0.005-0.1 M sodium phosphatebuffer, pH 6.0 containing 0.3-0.6 M sodium chloride in the next step.This fraction also contains other vitamin K dependent clotting factorssuch as clotting factor II (FII), clotting factor X (FX), lower amountsof clotting factor VII (FVII) together with residual amounts of IαIp.After dialysis to reduce osmolarity and salt concentration, the residualIαIp in the FIX fraction can be recovered by affinity chromatography onimmobilized heparin and step elution in buffers with increasing saltconcentration and osmolality. Fractions from an early elution step inthe wash buffer contain over 80% IαIp with very low FIX contamination.Elution of FIX occurs at a later step in a buffer with higher saltconcentration and osmolality. There is also a flow through fraction thatdoes not bind to heparin, is free of FIX and contains a mixture of IαIp(30-40%), vitamin K-dependent clotting factors, and thesolvent/detergent (S/D) used for virus inactivation. The S/D can beremoved in an additional chromatographic step of DEAE-Sepharose FF.

IαIp containing fractions collected from DEAE-Sepharose, immobilizedheparin or the flow through from heparin can be further purifiedindividually or as a pool by hydroxylapatite chromatography. Usingeither approach, the final preparation contains more than 90% ITI.

The concentrate purified using these protocols contains more than 90%IαI/PαI. It has been virus inactivated by solvent/detergent treatment.Terminal heating in final container with or without use of stabilizersfor more than 30 minutes or pasteurization at 55-65° C. in the presenceof stabilizers can be introduced as a second virus inactivation stepwithout significant loss of activity.

The resulting concentrate containing more than 90% IαI/PαI can be virusinactivated with S/D treatment or as a second inactivation step,terminal heating of the purified proteins with or without stabilizersfor 30 minutes or alternatively, pasteurization at 55-65° C. in thepresence of stabilizers.

A Strong Anion-Exchange Fraction

Instead of the eluate after solid-phase extraction with weakanion-exchanger DEAE Sephadex A50) an eluate after solid phaseextraction with a strong anion-exchanger Q Sephadex A50 can be used.Conditions for elution are described in German patent DE 4342132C1. Amixture of IαI and PαI do not bind or only weakly binds to themonolithic anion-exchange support DEAE-CIM. Other proteins such asclotting factors FII, FVII, FIX and FX, clotting inhibitors PC, PS andPZ, adhesion protein vitronectin and protease FSAP are eluted inseparate fractions. Further separations of the remaining contaminantscan be achieved in the next step using hydroxylapatite chromatography.

A Monolith Chromatographic Fraction

A DEAE CIM monolith (membrane) can be used for chromatographicseparation in FIX purification instead of DEAE Sepharose FF or otherparticle-based anion exchanger (See DE 4342132C1). Surprisingly, IαI andPαI did not bind or bound only weakly to the monolithic support. Otherproteins, such as FIX, vitronectin and FII, FVII (low amount) and FXwere eluted as separate fractions. The proteolytic activity, comingmainly from Factor VII Activating Protease (FSAP) (J. Roemisch,Biological Chemistry 383 (2002) 1119-1124) was also completely separatedin this purification step. Further separation of the remainingcontaminants from IαI/PαI containing fraction(s), namely traces ofvitamin K dependant clotting factors FII, FVII and FX were achieved inthe next step using hydroxylapatite chromatography.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted. Unless defined otherwise, all technical andscientific terms used herein have the meaning commonly understood by aperson skilled in the art to which this invention belongs. The followingreferences provide one of skill with a general definition of many of theterms used in this invention: Singleton et al., Dictionary ofMicrobiology and Molecular Biology (2nd ed. 1994); The CambridgeDictionary of Science and Technology (Walker ed., 1988); The Glossary ofGenetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); andHale & Marham, The Harper Collins Dictionary of Biology (1991).

INCORPORATION BY REFERENCE

The contents of all references (including literature references, issuedpatents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein in their entireties by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended with be encompassed by the following claims.

What is claimed is:
 1. A process for producing a blood plasma-derivedIαIp composition comprising a mixture of inter-alpha inhibitor protein(IαI) and pre-alpha protein (PαI), wherein the IαI and the PαI arepresent in said mixture in a physiological proportion, the processcomprising: isolating from blood plasma a plasma fraction containing IαIand PαI, wherein the IαI and PαI are present in a physiologicalproportion; and purifying the plasma fraction to obtain an IαIpcomposition with a purity of IαIp ranging from about 85% to about 100%pure, wherein the purifying comprises hydroxylapatite chromatography. 2.The process of claim 1, wherein the isolating comprises solid phaseextraction or chromatographing blood plasma.
 3. The process of claim 1,wherein the plasma fraction comprises a side fraction obtained from thepurification of clotting factor IX or from the purification of aprothrombin complex concentrate.
 4. The process of claim 1, wherein theplasma fraction is isolated as a cryosupernatant resulting fromcryoprecipitation of blood plasma.
 5. The process of claim 1, whereinthe plasma fraction is cryo-poor plasma.
 6. The process of claim 1,wherein the plasma fraction is human, primate, bovine, porcine, feline,or canine.
 7. The process of claim 1, further comprising obtainingblood, obtaining blood plasma, obtaining a side fraction obtained fromthe purification of clotting factor IX, obtaining a side fraction fromthe purification of a prothrombin complex concentrate, obtaining acryosupernatant resulting from cryoprecipitation of blood plasma orobtaining cryo-poor plasma.
 8. The process of claim 1, wherein thepurifying further comprises affinity chromatography.
 9. The process ofclaim 1, wherein the IαI and PαI present in the plasma fraction have anapparent molecular weight of between about 60,000 to about 280,000 kDa.10. The process of claim 1, further comprising: purifying the plasmafraction; virus inactivating the plasma fraction and/or the purifiedIαIp; the addition of stabilizers; pasteurization of the purified IαIp;or anion-exchange chromatography of the purified IαIp.
 11. The processof claim 10, wherein the further purifying the plasma fraction is bypassing through heparin affinity column and collecting the flow through(unbound) fraction; the virus inactivating is by a solvent/detergenttreatment or thermal inactivation; and the anion-exchange chromatographyof the purified IαIp is diethylaminoethyl (DEAE) Sepharose.
 12. Theprocess of claim 11, wherein the thermal inactivation comprisespasteurization at a temperature of between about 55 to about 65° C. ordry heat at 70 to 120° C. omega-amino acids, sugar, or combinationsthereof.
 13. A composition of IαIp comprising a mixture of inter-alphainhibitor protein (IαI) and pre-alpha protein (PαI), wherein the IαI andthe PαI are present in said mixture in a physiological proportion and:have a high trypsin inhibitory specific activity between about 1000 toabout 2000 IU/mg; have a half life of greater than one hour; comprise alight chain of inter-alpha inhibitor protein associated with at leastone of three heavy chains H1, H2 and H3; or comprise a light chain ofinter-alpha inhibitor protein associated with at least one of threeheavy chains H1, H2, H3 and H4.
 14. The composition of claim 13, whereintrypsin inhibitory specific activity is between about 1400 to about 2000IU/mg.
 15. The composition of claim 13, wherein the IαIp composition hasa half life of at least 5 hours.
 16. The composition of claim 13,wherein the IαIp composition has a half life of at least 10 hours.
 17. Acomposition of IαIp comprising a mixture of inter-alpha inhibitorprotein (IαI) and pre-alpha protein (PαI), wherein the IαI and the PαIare present in said mixture in a physiological proportion, saidcomposition having been prepared by the process according to claim 1.18. The composition of claim 17, further comprising an additionaltherapeutic agent.
 19. The composition of claim 18, wherein theadditional therapeutic agent is an anti-inflammatory agent, ananti-coagulant or an immunomodulator.
 20. A pharmaceutical compositioncomprising a therapeutically effective amount of the composition ofclaim 17, and a pharmaceutically acceptable carrier.
 21. A method oftreating an inflammation related disorder comprising administering to asubject in need thereof a therapeutically effective amount of thecomposition of claim 17, wherein the inflammation related disorder isselected from an acute inflammatory disease, sepsis, septic shock,rheumatoid arthritis, meningitis, Crohn's Disease, chronic obstructedpulmonary disease and rhinitis.
 22. The method of claim 21, wherein theIαIp is administered as a tablet, capsule, or injectables.
 23. A methodof treating an acute inflammatory disease, sepsis, septic shock,rheumatoid arthritis, meningitis, Crohn's Disease, chronic obstructedpulmonary disease and rhinitis in a subject, comprising: (a) determiningthe pre-treatment level of one or more of the following levels in asubject: (i) the level of IαI; (ii) the level of PαI; (iii) the level ofIαIp; (iv) the level of H3; (v) the level of H4; (vi) the level of H1;(vii) the level of H2; and (viii) the level of LC; and (b) administeringa therapeutically effective amount of the composition of claim 17 to thesubject.
 24. A method of monitoring the progress of a subject beingtreated with an IαIp therapy, comprising: (a) determining thepre-treatment level of one or more of the following levels, in asubject: (i) the level of IαI; (ii) the level of PαI; (iii) the level ofIαIp; (iv) the level of H3; (v) the level of H4; (vi) the level of H1;(vii) the level of H2; and (viii) the level of LC; (b) administering atherapeutically effective amount of the composition of claim 17 to thesubject; and (c) determining the level of one or more of the levels inthe subject after an initial period of treatment with the composition,wherein an increase of the level in the subject following treatment withthe composition indicates that the subject is likely to have a favorableclinical response to treatment with IαIp.
 25. A kit comprising acomposition according to claim 17 and instructions for therapeutic use.26. A method of treating sepsis or septic shock in a human in needthereof comprising administering to the human more than one dose of apharmaceutical composition comprising 1 to 50 milligrams (mg)inter-alpha inhibitor proteins (IαIps) per kilogram (kg) body weight ofthe human, wherein the IαIps comprise 5% to 95% by weight of thecomposition and comprise a mixture of 60% to 80% inter-alpha inhibitor(IαI) and 40% to 20% pre-alpha inhibitor (PαI), the composition issuitable for administration to the human, and each dose of thecomposition is administered every 4 to 120 hours from about 1 to about 6times per day.
 27. The method of claim 26, wherein said IαI and PαIcomprise a light chain (L) associated with at least one heavy chainselected from H1, H2, H3, and H4.
 28. The method of claim 27, whereinthe composition has a high trypsin inhibitory specific activity of 1000to 2000 IU/mg.
 29. The method of claim 28, wherein the trypsininhibitory specific activity of said composition is 1400 to 2000 IU/mg.30. The method of claim 26, wherein the composition has a half-life ofgreater than one hour.
 31. The method of claim 30, wherein thecomposition has a half-life of at least 5 hours.
 32. The method of claim31, wherein the composition has a half-life of at least 10 hours. 33.The method of claim 26, wherein the IαI and PαI present in saidcomposition have an apparent molecular weight of 60,000 to 280,000 Da.34. The method of 26 further comprising, prior to said administering,determining the pre-treatment level of one or more of the followingIαIps in the human: IαI, PαI, H3, H4, H1, H2, and LC.
 35. The method ofclaim 34 further comprising determining the level of one or more of IαI,PαI, H3, H4, H1, H2, and LC in the human after an initial period oftreatment with the composition.
 36. The method of claim 26, wherein thecomposition further comprises a stabilizer.
 37. The method of claim 26,wherein the composition is administered as a tablet, capsule, orinjectable.
 38. The method of claim 26, wherein the composition isadministered by intravenous infusion.
 39. The method of claim 26,wherein the composition is administered at least twice.
 40. The methodof claim 26, wherein the composition is administered until symptoms ofthe sepsis or septic shock improve.